There is increasing evidence for the existence of a family of tumor sup- pressor genes that are involved in different cancers. Using a technique known as microcell-mediated chromosome transfer, we mapped tumor suppressor genes involved in human and rodent cancers to chromosome 1 (endometrial cancer, fibrosarcomas), chromosome 3 (renal and lung cancer), chromosome 6 (endometrial cancer), chromosome 7 (choriocarcinoma), chromosome 9 (endo- metrial cancer), and chromosome 11 (cervical cancer, Wilms' tumor, rhab- domyosarcoma, lung cancer, fibrosarcoma) and chromosome 18 (endometrial cancer). Variants of chemically-immortalized Syrian hamster embryo cells were subcloned that had either retained (supB+) of lost (supB-) the ability to suppress tumorigenicity when hyridized with fibrosarcoma cell line. Both supB cell types are nontumorigenic; however, the supB-, but not supB+ cells exhibit conditional anchorage-independent growth potential. Examination of various cytoskeletal components of these cells revealed alterations of actin microfilament organization in supB- cells characteristic of tumor cells, while the supB+ cells exhibited microfilaments typical of normal fibroblasts in culture. To investigate the molecular basis for this cytoskeletal modification, total cellular proteins from the two cell types were compared by two-dimensional polyacrylamide gel electrophoresis. The most significant difference was reduction of the actin-binding protein tropomyosin in supB- cells. To examine the possibility of a direct relationship between TM-1 expression and the supB- phenotype, supB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. Antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage- independent growth potential that was indistinguishable from those charact- eristic of supB- cells. These data provide direct evidence that TM-1 can regulate microfilament organization and anchorage-independent growth.